Fig 2: Assessment of changes in chromosome morphology after induction of SMC2 depletion.For each cell clone, one metaphase plate is presented, illustrating the most common chromosome morphology without induction (aux–) and after induction (aux+). Also presented is a diagram summarizing the proportion of metaphase plates with uncondensed chromosomes, normal chromosome morphology and ambiguous. For each condition, the number of analyzed metaphase plates is indicated – N. a – HCT116-SMC2-mAC; cells with effective SMC2 depletion – positive control; b, c – cell clones tested.

Fig 3: Effects of pH on AtAFB2 and OsTIR1 AID degradation efficiency. (A) High pH levels inhibit auxin-induced eGFP degradation in HAP1 clones with RAD21 and SMC2 modifications. Experiments were performed in duplicate. (B) IAA and NAA auxin analogs show similar sensitivity to pH changes (HAP1 clones). Experiments were performed in duplicate. (C) eGFP levels in the mosaic HAP1 clone SMC2-miniIAA7-eGFP. Blue color indicates eGFP levels in Cherrypos subpopulation, and purple color indicates Cherryneg subpopulation. The shift between two peaks illustrates minor basal degradation induced by AtAFB2 in Cherrypos cells. (D) Shift of mES cell population (OsTIR1 system) toward left on the FITC-A axis in acidic media.

Fig 4: Comparison of degradation efficiency with AFB protein driven by the same EF1a promoter. (A) Scheme of genetic constructs used. (B) Dynamics of POI depletion in mESC clones (Rad21 colored in red; Smc2 colored in blue). Vectors expressing AFB proteins: pSH-EFIRES-P-OsTIR1-mCherry-weak NLS and pSH-EFIRES-P-AtAFB2-mCherry-weak NLS, were randomly integrated. For this analysis, all clones were grown in puromycin, and the proportion of Cherry-positive cells in each of them was more than 95%. For each clone, the measurement was carried out twice.

Fig 5: Degradation efficiency and dynamics of POI depletion in mESC and HAP1 clones with randomly integrated OsTIR1 and AtAFB2 constructs. POI degradation efficiency in mESC clones (A) and HAP1 clones (B). Each dot represents one independent cell clone derived after AFB protein gene integration. For each clone, the measurement was carried out once. The black horizontal line shows the median of the group. (C) POI degradation dynamics for selected mESC clones (Rad21-mAID-Clover, Smc2-mAID-Clover, Rad21-miniIAA7-eGFP, Smc2-miniIAA7-eGFP) with OsTIR1 and AtAFB2. For each clone, the experiment was carried out once. (D) POI degradation dynamics for HAP1 clones (RAD21-miniIAA7-eGFP, RAD21-mAID-Clover) with AtAFB2 and OsTIR1. For each clone, the experiment was carried out once. (E) POI degradation dynamics for HAP1 clones (SMC2-miniIAA7-eGFP, SMC2-mAID-Clover) with AtAFB2 and OsTIR1. Numbers in brackets are individual clones’ designations. For each clone, the experiment was carried out once.